Bordetella pertussis, the etiologic agent of whooping cough, is a substantial cause of morbidity and infant mortality worldwide. The majority of the estimated annual 279,000 infant pertussis deaths occur in developing countries (WHO, 2005). In the United States, 25,616 cases of pertussis were reported during 2005 (Kretsinger et al., MMWR Recomm. Rep. 55:1-44, 2006). The three most common Bordetella human upper respiratory pathogens are B. pertussis, B. parapertussis, and B. holmesii. Others of the nine known species include Bordetella bronchiseptica, Bordetella hinzii, and Bordetella trematum. The B. parapertussis infection is less severe than B. pertussis, with symptoms that include a paroxysmal cough and a whoop that, if present, persists for a shorter duration (Mattoo and Cherry, Clin. Microbiol. Rev. 18:326-382, 2005). B. holmesii was first associated with septicemia in immunocompromised patients (Weyant et al., J. Clin. Microbiol. 33:1-7, 1995) and has been recovered from nasopharyngeal specimens of patients with pertussis-like diseases in Massachusetts (Yih et al., Emerg. Infect. Dis. 5:441-443, 1999). Therefore, differentiating these three species is essential to providing an accurate diagnosis of pertussis-like diseases or whooping cough in patient populations.
Prior art diagnostic methods to detect B. pertussis include culture, singleplex real-time polymerase chain reaction (PCR), and serology. Culture is 100% specific; however, it suffers from low sensitivity (12-60%) and lengthy incubation periods from 5 to 10 days (Wendelboe and Van Rie, Expert Rev. Mol. Diagn. 6:857-864, 2006). The sensitivity of culture is highest in young infants tested after a short duration of symptoms and the lowest in adolescents and adults tested after a longer duration of symptoms (Riffelmann et al., J. Clin. Microbiol. 43:4925-4929, 2005).
In the United States, the number of reported adult and adolescent pertussis cases has substantially increased in the last two decades; however, most cases are not laboratory confirmed, highlighting the need for additional diagnostic tests with higher sensitivity than culture. Although the lengthy incubation period for culture makes it impractical as a rapid diagnostic test, culture in combination with PCR is a reliable option for case detection during a public health response to a pertussis outbreak (Sotir et al., Clin. Infect. Dis. 44:1216-1219, 2007). Serologic assays are reviewed in detail elsewhere (Broder et al., 2006; Centers for Disease Control and Prevention [CDC], MMWR 56:837-842, 2007; Fry et al., J. Med. Microbiol. 53:519-525, 2004; Kretsinger et al., 2006; Mattoo and Cherry, 2005; Sotir et al., 2007).
Real-time PCR assays, which can be rapid (2-24 h), specific (86-100%), and sensitive (70-99%), have been designed to detect B. pertussis (Wendelboe and Van Rie, 2006). Conventional PCR assays introduced in 1989 (Houard et al., Res. Microbiol. 140:477-487, 1989) for the detection of B. pertussis are being replaced by real-time PCR assays in clinical diagnostics. However, standardizing these PCR methods is challenging (Arber, FEMS Microbiol. Rev. 24:1-7, 2000; Broder et al., MMWR Recomm. Rep., 55:1-50, 2006; CDC, MMWR 53:216-219, 2004; Douglas et al., J Med Microbiol 38:140-144, 1993; Fry et al., 2004). Several chromosomal regions have been used as targets, such as adenylate cyclase (Douglas et al., 1993), pertactin (Byrne and Slack, BMC Infect. Dis. 6:53-61, 2006; Makinen et al., Emerg. Infect. Dis. 7:952-958, 2001; Vincart et al., J. Med. Microbiol. 42:847-849, 2007), porin (Li et al., J. Clin. Microbiol. 32:783-789, 1994; Qin et al., J. Clin. Microbiol. 45:506-511, 2007), recA (Antila et al., J. Med. Microbiol. 55:1043-1051, 2006; Qin et al., 2007; Vielemeyer et al., J. Clin. Microbiol. 42:847-849, 2004), pertussis toxin promoter region (Houard et al., Res. Microbiol. 140:477-487, 1989; Knorr et al., BMC Infec. Dis. 6:62-74, 2006; Makinen et al., 2001; Nygren et al., J. Clin. Microbiol. 38:55-60, 2000; Reizenstein, Dev. Biol. Stand. 89:247-254, 1997; Stefanelli et al., Diag. Microbiol. Infect. Dis. 24:197-200, 1996), and, most frequently, insertion sequence IS481 (Glare et al., J. Clin. Microbiol. 28:1982-1987, 1990; McPheat and McNally, J. Gen. Microbiol. 133:323-330, 1987a, FEMS Microbiol. Lett. 41:357-360, 1987b; Templeton et al., J. Clin. Microbiol. 41:4121-4126, 2003; van der Zee et al., J. Clin. Microbiol. 31:2134-2140, 1993).
Accurate identification of pertussis cases in respiratory disease outbreaks is of particular importance because some interventions, such as vaccination, are pathogen specific. PCR confirmation is included in the case definition of pertussis from the Council of State and Territorial Epidemiologists. A confirmed case of pertussis is any person with acute cough illness of any duration with isolation of B. pertussis, or a case that meets the clinical case definition and is confirmed by PCR, or a case that meets the clinical definition and is epidemiologically linked directly to a case confirmed by either culture or PCR (Broder et al., 2006; Kretsinger et al., 2006). However, in respiratory disease outbreaks, positive results with a single PCR assay targeting the insertion sequence IS481 have led to a false diagnosis of pertussis, demonstrating the need for different or additional target (CDC, 2007; Farrell et al., J. Clin. Microbiol. 38:4499-4502, 2000; Lievano et al., J. Clin. Microbiol. 40:2801-2805, 2002; Muyldermans et al., J. Clin. Microbiol. 43:30-35, 2005).
Thus, there is a need for a rapid, sensitive, and discriminatory assay for detection of Bordetella sp. in complex clinical or laboratory samples in the presence or absence of other bacterial or viral agents.